The proposed research is a biochemical-genetic study of mitochondrial rRNA splicing in Neurospora. The gene encoding the mitochondrial large (25 S) rRNA of Neurospora contains an intervening sequence of ca. 2.3 kb. In previous work, we identified two allelic, nuclear mutants (299-9 and 286-67) which have a temperature-sensitive defect in splicing this RNA. When grown at the nonpermissive temperature (37 degrees), the mutants accumulate a 35 S pre-rRNA which is a colinear transcript of the 25 s RNA gene including the intervening sequence. We have now identified a new nuclear mutant (AEG-193) which also accumulates 35 S RNA, but which is not allelic with the previously studied mutants. Specific aims of the proposed research are: (a) to continue to characterize the mutants, (b) to continue to isolate and characterize revertants of mutants, (c) to characterize the tertiary structure of 35 S RNA and 35 S RNA-ribonucleoprotein particles, and (d) to develop techniques for assay purification of the RNA splicing enzymes. These studies are intended to provide insight into the mechanism and physiological role of RNA splicing which should be relevant to all eukaryotic cells.